The nuclear-factor ?B (NF-?B) family of transcription factors plays a central role in regulating many physiological processes including innate and adaptive immunity. In addition, accumulating evidence suggests that inappropriate activation of NF-?B occurs in many types of human cancers. Although genetic alterations of specific core components of the NF-?B signaling pathway have been identified in a few lymphoid malignancies, the molecular alterations that lead to and the consequences of increased NF-:B activity in the pathogenesis of human cancers remain incompletely understood. In recent work, we have identified the inhibitor of ?B kinase 5 (IKBKE, IKKi IKK5) as amplified and overexpressed in a substantial subset of human breast cancer cell lines and tumors. IKK5 is required for the survival of breast cancer cell lines that harbor IKK5 copy number gain and confers a tumorigenic phenotype when expressed in immortalized human mammary epithelial cells. Breast cancer cell lines and tumors that overexpress IKK5 exhibit increased NF-?B activity, which is essential for their transformed phenotype. These observations classify IKK5 as a breast cancer oncogene and may also explain the increased NF-?B activity previously described in a subset of human breast cancers. Based on these observations, this proposal focuses on investigating the role of IKK5 in breast cancer pathogenesis. Specifically, biochemical, genetic and molecular biological approaches will be applied to identify and characterize which substrates and interacting proteins of this non-canonical IKK are critical for tumorigenicity, to investigate how ubiquitination regulates IKK5 activity and function in breast cancer and to dissect the roles of IKK5 in tumor initiation and maintenance in genetically engineered mice. Investigating the regulation and function of IKK5 in breast cancer development will not only enhance our mechanistic understanding of this non-canonical IKK regulator but will also clarify the role of NF-?B signaling in the development of human epithelial cancers. In addition, these studies will provide a foundation for strategies to target this kinase oncogene therapeutically.